Solving a Sticky Problem: Understanding Enzyme Binding to Lignocellulosic Biomass during Biofuel Production

解决棘手问题：了解生物燃料生产过程中酶与木质纤维素生物质的结合

• 批准号: 1236120
• 负责人: Tim Whitehead
• 金额: $ 29.98万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1236120&HistoricalAwards=false
• 关键词: Solving; Sticky; Problem; Understanding; Enzyme

PI: Whitehead, TimProposal Number: 1236120Institution: Michigan State UniversityTitle: Solving a Sticky Problem: Understanding Enzyme Binding to Lignocellulosic Biomass during Biofuel ProductionBiological-mediated conversion of cellulosic biomass to useful fuels and chemicals is a promising avenue towards energy sustainability. A critical impediment for this avenue is the cost of the cellulase enzymes needed to deconstruct biomass to fermentable sugars. At the current cellulase-to-substrate loading the cost per gallon of fuel produced from lignocellulose would still be at least an order of magnitude higher than the enzymes required to degrade starch, even if the enzymes could be produced as cheaply as soy protein. One strategy to reduce enzyme requirements is to recycle the cellulases in a continuous process, but enzyme inactivation during biomass saccharification severely limits this approach. PI?s recent findings show that enzyme inactivation occurs through non-productive interactions with residual lignin (i.e. the cellulases adsorb to hydrophobic lignin surfaces hastening their denaturation) for most pretreatment chemistries and biomass sources.The PI hypothesizes that minimizing the potential of mean force between the cellulases and lignin polymer will reduce surface adsorption between the two, hence minimizing enzyme inactivation. Thus, the primary objective of this project is to test the hypothesis that selective variation of cellulase surface chemistry can decrease lignin-mediated inactivation due to reduced protein surface adsorption to lignin. The hypothesis will be tested by using computational protein design to identify mutations that result in different enzyme surface potentials predicted to minimize enzyme-lignin adsorption, and then genetically program these mutations in two representative cellulases from the workhorse microbe Trichoderma reesei. A large number (ca. 1000) of cellulase variants will be recombinantly expressed and screened for solubility and activity on cellulosic substrates. Surviving variants will be tested for retention of structure, solubility, and stability. Adsorption isotherms will be recorded for the variants for natural and synthetic lignin of varying surface chemistry and microstructure. The PI will test this hypothesis for cellulase catalytic domains, carbohydrate binding modules, and the full-length cellulases.The project will provide fundamental quantitative understanding of protein-surface adsorption effects with significant implications for commercial-scale bioenergy production using lignocellulosic biomass. More generally, this work will supply datasets for identifying critical parameters governing enzyme-surface interactions crucial for disparate applications such as separations of single wall carbon nanotubes, laundry detergents, biomaterials for medical uses and immobilized biocatalysts.This project will engage students from K-12, undergraduate, and graduate levels from various interdisciplinary programs and under-represented groups to broaden their understanding of the role of industrial biotechnology in mitigating bioenergy related problems in an environmentally sustainable manner. The project will utilize the established outreach educational programs at the Great Lakes Bioenergy Research Center to create bioenergy-relevant educational materials for science teachers within the greater mid-Michigan school community.

主要研究者：Whitehead，TimProposal编号：1236120机构：密歇根州立大学标题：解决一个棘手的问题：了解酶结合到木质纤维素生物质在生物燃料生产生物介导的纤维素生物质转化为有用的燃料和化学品是一个有前途的途径，实现能源的可持续性。这一途径的关键障碍是将生物质解构为可发酵糖所需的纤维素酶的成本。在目前的纤维素酶-底物负荷下，从木质纤维素生产的每加仑燃料的成本仍然比降解淀粉所需的酶高至少一个数量级，即使酶可以像大豆蛋白一样便宜地生产。减少酶需求的一种策略是在连续过程中回收纤维素酶，但生物质糖化过程中的酶失活严重限制了这种方法。派？最近的研究结果表明，对于大多数预处理化学品和生物质来源，酶失活是通过与残余木质素的非生产性相互作用（即纤维素酶吸附到疏水木质素表面，加速其变性）发生的。PI假设最小化纤维素酶和木质素聚合物之间的平均力的潜力将减少两者之间的表面吸附，从而最小化酶失活。因此，本项目的主要目的是测试的假设，纤维素酶表面化学的选择性变化可以减少木质素介导的失活，由于减少蛋白质表面吸附到木质素。该假设将通过使用计算蛋白质设计来验证，以确定导致不同酶表面电位的突变，预测这些突变将使酶-木质素吸附最小化，然后在来自主力微生物里氏木霉的两种代表性纤维素酶中对这些突变进行遗传编程。一个很大的数字（CA）。1000）的纤维素酶变体进行重组表达，并筛选其在纤维素底物上的溶解性和活性。将检测存活变体的结构保留、溶解度和稳定性。将记录不同表面化学和微观结构的天然和合成木质素变体的吸附等温线。PI将测试纤维素酶催化结构域，碳水化合物结合模块，和全长纤维素酶的假设。该项目将提供基本的定量了解蛋白质表面吸附的影响，具有重要意义的商业规模的生物能源生产使用木质纤维素生物质。更一般地说，这项工作将提供数据集，用于确定决定酶-表面相互作用的关键参数，这些参数对不同的应用至关重要，例如单壁碳纳米管的分离，洗衣剂，医用生物材料和固定化生物催化剂。和研究生水平，从各种跨学科的计划和下，代表团参加了一次讲习班，以扩大他们对工业生物技术在以环境可持续方式缓解生物能源相关问题方面的作用的理解。该项目将利用五大湖生物能源研究中心已建立的外展教育计划，为密歇根州中部学校社区的科学教师制作生物能源相关的教育材料。